Systems and methods for measurement optimization

ABSTRACT

The invention relates to methods and systems for detecting and or analyzing an agent in a sample with a chip having optical components incorporated therein.

RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.11/250,179, filed Oct. 13, 2005, which claims the benefit of U.S.Provisional Application Ser. No. 60/618,453 entitled “MeasurementOptimization Using Microlens” filed Oct. 13, 2004, the entire contentsof each are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates in part to a chip incorporating a microfluidicchannel and a lens used to detect contents of the channel.

BACKGROUND OF THE INVENTION

Systems are known that utilize chips to receive a sample for opticalinspection to determine whether an agent is present in the sample. Theagents often include polymers, such as nucleic acids with detachablylabeled probes bound thereto in a particular manner. The sample ispassed through a detection zone where an excitation signal illuminatesthe probe. The labels of the probe are excited when present in thedetection zone and emit an emission signal. The emission signal isreceived by a collector that is separate from the chip and that, inturn, directs the emission signal to a detector as a portion of adetection signal. The characteristics of the emission signal relative tothe sample, the excitation signal, the surroundings, and/or othercharacteristics are then used by the system to detect the presence ofthe polymer and/or to analyze structure of the polymer.

Alignment between optical components and chips of prior art systems canbe critical to the reliability of the system and the quality ofinformation produced by the system. Emitters and/or collectors are oftenrequired to be focused into a small detection zone so that the positionof a probe associated with a polymer can be determined with precisionwhen present in the detection zone. Expensive, adjustable focus lensesare used in prior art systems to accommodate variations in thepositional relationship between a chip used by the system and opticalcomponents that are separate from the chip. Costly motion controlsystems are also used in prior art systems to maintain the positionalrelationship between optical components and/or to minimize the impact ofmovement, such as vibrations in the system. There exists a need in theart for a system that minimizes the costs associated with positioning achip relative to optical detection components and is less (if at all)sensitive to vibrations.

Prior art detection systems can have difficulty distinguishing emissionsignals from noise and/or disturbances within the system. This may bethe case particularly in systems that attempt to detect a single polymeror molecule. Noise and/or disturbances may emanate from any number ofsources, including emission signals that are overlapped with excitationsignals in the system prior to being received by a detector. To thisend, there also exists a need for detection and analysis systems thatminimize or prevent the overlap of emission and excitation signals.There is also a need to increase the proportion of a signal receivedfrom each agent relative to background noise, transmitted illumination,and/or path fluorescence, particularly for single molecule detection.Compound lenses with relatively high numerical apertures are used inprior art systems to increase the proportion of signals received fromany given agent. However, such compound lenses are often expensive andrequire significant amounts of space within a system. This prevents suchhigh numerical aperture lenses from being incorporated in many detectionsystems, particularly where multiple detection zones are desired.Consequently, there is a need in the art for high numerical lens capableof being incorporated into a system with smaller spatial requirementsand at a lower cost.

Expensive and space consuming lenses preclude the utilization ofmultiple detection zones on a single chip in prior art systems. In thisregard, the amount of information that can be obtained from a sample ina given amount of time (i.e., throughput) can be limited in prior artsystems. There is a need to reduce the cost and size of opticalcomponents in detection systems such that higher throughput, parallelprocessing of samples on a chip can be achieved in a cost effectivemanner.

SUMMARY OF THE INVENTION

The invention provides, in its broadest sense, a system capable of rapidand reliable detection and/or analysis of agents, such as biohazardousagents. The system combines various technologies, includingmicrofluidics and single molecule detection capability. In someembodiments, microfluidic channels and optics are incorporated directlyinto a chip, such that the optics can be produced in-focus and at alower cost. Incorporating optics directly into a chip can reduce theimpact that vibrations have on the reliability of information producedby the system. Incorporating optics into the chip can also allownumerous detection zones to be placed into a single chip, thusincreasing the amount of information that can be derived from a samplein the chip during a given time frame.

In one aspect, the invention provides a chip for use in detecting anagent. The chip comprises a microfluidic channel incorporated into thechip. The microfluidic channel is adapted to deliver a fluid that maycontain an agent to a detection zone that lies at least partially in thechannel. An illuminator is incorporated into the chip and is adapted todirect an excitation signal to the detection zone. A concave reflectoris incorporated into the chip and has a focal point at the detectionzone. The concave reflector is constructed and arranged to receive anemission signal from the agent when present in the detection zone and toreflect the emissions signal to a detector.

In one aspect, the invention provides a chip for use in detecting anagent. The chip comprises a microfluidic channel incorporated into thechip. The microfluidic channel is adapted to deliver a fluid that maycontain an agent to a detection zone that lies at least partially in thechannel. The chip also comprises a concave reflector incorporated intothe chip in fixed relationship with respect to the channel. Thereflector has a focal point at the detection zone and the concavereflector is constructed and arranged to receive an emission signal fromthe agent when present in the detection zone and to reflect the emissionsignal to a detector.

The invention further provides a method that comprises providing a chipas described herein. The method also comprises providing a fluid thatmay contain the agent to the channel and illuminating the detection zonewith the excitation signal to cause any agent present in the detectionzone to emit an emission signal. The method further comprises receivingthe emission signal with the concave reflector and reflecting theemission signal toward the detector to determine whether the agent ispresent in the detection zone.

In still another aspect, the invention provides a chip for use indetecting an agent. The chip comprises a microfluidic channelincorporated into the chip. The microfluidic channel is adapted todeliver a fluid containing an agent to a plurality of detection zonesthat each lie at least partially in the channel. A plurality of concavereflectors are incorporated into the chip and are each held in a fixedrelationship with respect to one of the plurality of detection zones. Aplurality of illuminators are incorporated into the chip, each of theplurality of illuminators are adapted to provide an excitation signal toone of the plurality of detection zones.

Various embodiments apply equally to the various aspects of theinvention and for the sake of convenience these are recited once below.

In one embodiment, the chip includes a plurality of concave reflectorsand illuminators each associated with one of a plurality of detectionzones at the channel. In one embodiment, fluid is provided that maycontain the agent comprises providing the fluid to each of the pluralityof detection zones.

In one embodiment, the concave reflector is incorporated into the chipin a fixed relationship with respect to the channel.

In another embodiment, a solid medium provides a pathway from themicrofluidic channel to the concave reflector along which the emissionsignal can travel without substantial refraction. In one embodiment, thesolid medium extends from a wall of the channel to the concavereflector. Still, in one embodiment, a cover slip is adapted to matewith the chip to enclose the channel and the concave reflector isincorporated into the cover slip. In still another embodiment, the solidmedium provides a pathway from the microfluidic channel to theilluminator along which the excitation signal can travel withoutsubstantial refraction.

In one embodiment, the illuminator and the concave reflector are onopposed sides of the channel. In one embodiment, the illuminator is arefractive illuminator having a focal point substantially located at thefocal point of the concave reflector. In another embodiment, theilluminator is a reflective illuminator having a focal pointsubstantially located at the focal point of the concave reflector. Instill another embodiment, the concave reflector includes an aperturepositioned to allow the excitation signal to pass therethrough toprevent the excitation signal from being reflected toward the detector.In yet a further embodiment, the concave reflector is constructed andarranged to reflect at least a portion of the excitation signal.

One embodiment includes a wavelength specific filter positioned toreceive the emission signal and the excitation signal from the concavereflector. The wavelength specific filter is adapted to direct at leasta portion of the emission signal to the detector and to prevent theexcitation signal from reaching the detector. In some embodiments, asecond reflector is constructed and arranged to receive and reflect theemission signal to the detector while allowing the excitation signal topass thereby.

In one embodiment, the illuminator includes a refractive illuminatorhaving a focal point substantially located at the focal point of theconcave reflector and the illuminator and the concave reflector arepositioned on a common side of the channel. In one of such embodiments,the illuminator and the concave reflector are constructed and arrangedsuch that overlap of the excitation signal and the emission signal isminimized. The concave reflector can include a central aperture. Therefractive illuminator can be positioned at the central aperture. Theembodiment can include a second reflector constructed and arranged toreflect the excitation signal back through the aperture and toward therefractive illuminator. The second reflector can be substantiallylocated at the focal point of the concave reflector.

In some embodiments, the concave reflector has a collection half anglegreater than 50 degrees, greater than 65 degrees, or greater than 85degrees. In some embodiments, the reflector has a numerical aperture of1.0 or greater, or of 1.3 or greater.

In one embodiment, the reflector is a parabolic reflector. In oneembodiment, the illuminator includes a waveguide incorporated into thechip. In one embodiment, a waveguide can be constructed and arranged toilluminate the microfluidic channel with an evanescent excitationsignal.

In some embodiments, the chip comprises a plurality of pairs of concavereflectors and illuminators, each pair associated with a correspondingdetection zone. The chip can comprise more than 50 pairs of concavereflectors and illuminators. The plurality of pairs of concavereflectors and illuminators can be arranged in serial along a commonchannel. The chip can also comprise a plurality of channels, with theplurality of pairs of concave reflectors and illuminators arranged aboutthe plurality of channels.

In some embodiments, the detection zone is circular in shape. Thedetection zone can have a diameter of about 1.7 microns. The detectionzone can be elliptical in shape, and can have a minor diameter of about1.7 microns. In some embodiments, the detection zone extends beyond thechannel.

In some embodiments, the agent comprises a plurality of agents. Theagent can be a polymer. The polymer can be a nucleic acid optionallyselected from the group consisting of DNA or RNA. The polymer can be apeptide including a protein. The agent can also be a cell or a pathogen

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments and to the accompanyingdrawings.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

The phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use herein of“including”, “comprising”, “having”, “containing”, “involving”, andvariations thereof, is meant to encompass the items listed thereafterand equivalents thereof as well as additional items.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying Figures are not intended to be drawn to scale. Forpurposes of clarity, not every component may be labeled in every Figure.

FIG. 1 is a cross sectional view of optical components incorporated intoa chip.

FIG. 2 is a schematic view of components commonly found in systems thatmate with chips of the present invention.

FIG. 3 is a cross sectional view of a chip formed of separable opticalcomponents that are incorporated into a chip.

FIG. 4 is a cross sectional view of a chip with a refractive illuminatorand a reflective collector located on opposed sides of the chip.

FIG. 5 is a cross sectional view of a chip with a reflective illuminatorand a reflective collector located on opposed sides of the chip.

FIG. 6 is a perspective view of a chip with a plurality of pairs ofcollectors and illuminator pairs arranged in series along the length ofa channel.

FIG. 7 is a perspective view of a chip with a plurality of pairs ofcollectors and illuminators arranged in parallel along a plurality ofchannels.

FIG. 8 is a cross sectional view of a waveguide incorporated into achip.

It is to be understood that the Figures are not required for enablementof the invention.

Other aspects, embodiments and features of the invention will becomeapparent from the following detailed description of the invention whenconsidered in conjunction with the accompanying Figures.

All patent applications and patents cited herein incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides systems and methods for detecting whether anagent is present in a sample. The systems and method generally use chipshaving microfluidic channels incorporated therein. Optical componentsare incorporated into the chip to provide various benefits over priorart systems with components that are separate from the chip. In someembodiments, one or more reflectors and/or illuminators are incorporatedinto the chip during manufacturing to reduce overall system cost.Incorporating illuminators and/or reflectors into the chip can eliminatethe need for costly optical components in the system—like adjustableobjective lenses. Moreover, incorporating optical components into thechip can make the system less susceptible to vibrations and/or provide achip that is manufactured in focus. The optical components can bearranged to maximize their numerical aperture and thus improve thesignal to noise ratio of emissions received by the system. Still, insome embodiments overlap between the excitation and emissions signal isminimized to increase the signal to noise ratio. Optical components canalso be incorporated into the chip in arrays to increase the amount ofinformation derived from a given sample as the sample passes throughchannels in the chip.

As used herein, the term “emission signal” denotes emissions of an agentin the detection zone, such as the fluorescent emissions of some labelsthat may be incorporated into an agent. The term “detection signal” isused to denote an entire signal received from the detection zone of asystem, regardless of how many emission signals it may contain and/orthe amount and type of noise or disturbances that it may also contain.In this manner, the detection zone and its contents define the detectionsignal. As used herein, the term “excitation signal” is used to describethe emissions of an emitter that may be used to excite a label. As usedherein, the term “detection zone” is used to denote the zone that isilluminated at a chip by an excitation signal in the system. In someembodiments, the detection zone has a circular cross section across atthe fluidic channel and is further defined by the intersection of acircular illumination field and the channel. The circular detection zonecan have a diameter of 1.7 microns. The detection zone can be anelliptical section across the fluidic channel defined by theintersection of an elliptical illumination field and the channel, or canextend beyond the channel. The elliptical detection zone can have minoraxes of 1.7 microns. In some embodiments, the detection zone is definedby the reach of an evanescent excitation signal that impinges upon amicrofluidic channel.

As used herein, the term “collector” denotes an optical component thatreceives and re-directs optical signals from a detection zone. In manyembodiments, the collector includes a concave reflector with a focalpoint at the detection zone. The collector has a parabolic shape in someembodiments, but can have different shapes to suit differentapplications. In some embodiments, the shape of the collector isadjusted to accommodate refraction that may occur to signals between thedetection zone and the collector.

As used herein, the term “illuminator” denotes an optical component thatdirects an excitation signal to a detection zone. In some embodiments,the illuminator is a refractive illuminator with a curved surface thatreceives an excitation signal from an emitter like a laser source andfocuses the excitation signal to a focal point. In other embodiments,the illuminator comprises a concave, reflective surface that focuses anexcitation signal to a focal point. A waveguide incorporated into thechip is one example of such an illuminator.

Turn now to FIG. 1, which shows optical components incorporated into achip 20 according to one embodiment. The chip includes a microfluidicchannel 22 adapted to receive a sample 24 comprising a carrier fluidthat may contain an agent 26. An illuminator 28 is positioned on oneside of the channel to receive an excitation signal 30 from the systemand to focus the excitation signal at the channel. A concave, reflectivecollector 32 is positioned to have a focal point that lies at thechannel, coincident with the focal point of the illuminator. Thecollector receives a detection signal from the detection zone andreflects the detection signal toward a downstream detector 34 (notshown) for analysis. The detection signal includes emission signals 36from any agents that are illuminated by the excitation signal whenpassing through the detection zone 38.

FIG. 1 also illustrates the path followed by the excitation and emissionsignals through the optical components of the chip, according to oneembodiment of the invention. The refractive illuminator receives anexcitation signal from an emitter and focuses the excitation signalthrough an aperture 40 in the collector to a focal point at the channel.The focused excitation signal defines a detection zone at the channel. Aconcave mirror 42 positioned on the opposite side of the chip reflectsthe excitation signal at a lower irradiance back through the chip andthrough the aperture. In another embodiment, the mirror reflects theexcitation signal transversely out of the chip. Agents or otherexcitable components present in the detection zone emit emission signalswhen illuminated by the excitation signal. A concave reflector, having afocal point coincident with the focal point of the illuminator, receivesa detection signal from the detection zone that includes the emissionsignals from any agents in the detection zone. Either directly orindirectly via intermediate optical components 44 (not shown) such asmirrors and/or filters, the detection signal is then reflected by thecollector toward a downstream detector.

FIG. 2 shows components found in many systems 21 that may interface withchips of the present invention. The illustrated system includes aplurality of emitters 46, such as lasers, to create excitation signalsthat are directed toward the illuminators of the chip. The system alsoincludes a plurality of detectors 34 that receive detection signals fromthe chip. The detectors convert detection signals into electronicsignals that are analyzed by downstream data processors. The dataprocessors determine the presence and/or structure of agents in samplesprovided to the chip. Some examples of detectors that may be used indetection systems include avalanche photo diodes, and channelphotomultiplier tubes. The illustrated detection system includes asample introduction port 48 that receives a sample to be delivered tothe microfluidic channel of the chip. The sample is moved throughplumbing in the system and into a port on the chip by a pump or throughelectrokinetics. Once received in the port, the sample is deliveredthrough the channels in the chip and through detection zones positionedat points of the channels. The sample is then delivered through an exitport 50 of the chip and into a waste collection reservoir of the system.

In one embodiment, a field mirror is used to increase the illuminationat the detection zone. The excitation signal is reflected off of themirror and back through the detection zone toward the illuminator. Inthis sense, the excitation signal passes through the detection zonetwice, increasing the illumination at the detection zone by a factor oftwo. It is to be appreciated that not all embodiments of the inventioninclude field mirrors, and those that do include a field mirror are notrequired to reflect light back toward the illuminator. In someembodiments, the field mirror comprises a flat surface formed of goldthat is embedded into the chip. In some embodiments, the mirror isincorporated directly into the chip at the bottom of the channel. Inother embodiments, the field mirror comprises a reflective ellipsoidalsurface embedded into the chip. Some embodiments may have a field mirroror a prism that directs the excitation signal out of the chip in atransverse direction, rather than back toward the illuminator. Still, inother embodiments, the excitation signal may pass through the chip fromone side to another without being reflected, as aspects of the inventiondo not require a field mirror. In other embodiments, a beam dump isplaced adjacent to the chip, or is incorporated into the chip to receivethe excitation signal after it has passed through the chip.

In one embodiment of FIG. 1, the collector includes a central aperturethat receives the illuminator. The aperture is positioned in thecollector to allow transmission of the excitation signal to thedetection zone. The aperture of the collector may also allow exit of theillumination in direction away from the detector. Portions of emissionsignals directed toward the aperture will not be reflected toward thedetector. However, the loss of such emission signals can be offset byelimination of the excitation signal from the detection signal. Althoughthe aperture in FIG. 1 is located centrally within the reflector, it canbe located elsewhere in other embodiments. By way of example, theaperture can be positioned on a side of the reflector. In otherembodiments, the collector may lack an aperture altogether, as aspectsof the invention are not limited in this respect.

The collector, illuminator, and aperture of FIG. 1 are constructed andarranged to prevent portions of the detection signal that have beenoverlapped with the excitation signal from being received by thecollector. As is shown by the signal tracings in FIG. 1, the excitationsignal reaches the detection zone without crossing the path of anyemission signals that are eventually directed toward a detector in thesystem. In this sense, overlap between the excitation and emissionsignals is avoided, which can decrease the signal to noise ratio of thesystem by increasing background fluorescence in the detection signal.Some overlap between the excitation signal and the emission signal mayoccur directly above the detection zone. However these portions of theemission signals are not likely to pose problems as they typically willnot be reflected by the collector but rather allowed to pass through theaperture in the collector and out of the chip. It is to be appreciatedthat not all embodiments of the invention minimize overlap betweenexcitation and emission signals, and that overlap is acceptable in someembodiments. For instance, the excitation signal, once reflected by theconcave mirror 42, has less irradiance and should not adversely affectthe emission signals if overlap occurs.

The excitation signal can be utilized by the system to determine whenthe chip is in proper alignment. By way of example, in one embodiment ofFIG. 1, the field mirror is sized and positioned to reflect theexcitation signal through the central aperture in the collector onlyonce the excitation signal is in position at the channel. In someembodiments, the bottom of the channel may serve to reflect theexcitation signal itself, or may include a reflective coating or mirrorto help accomplish this effect. The system includes a detector thatreceives the excitation signal after it is reflected from the mirror toindicate that the chip is positioned properly. In other embodiments, thefield mirror or a prism may direct the excitation signal toward adetector in a different direction, such as transversely out of the chip,to determine proper positioning. Still, some embodiments may lack afield mirror that reflects the excitation signal. In such systems, adetector can be placed on a side of the chip that is opposed to theilluminator, such that the excitation passes through the aperture,across the chip and into a detector when the chip is positionedproperly. Still, other embodiments can have other features to indicateproper positioning of the chip. Some features include switches thatcontact portions of the chip to indicate proper position, as aspects ofthe present invention are not limited to any one particular mode ofdetermining proper chip positioning, and some embodiments completelylack such a capability.

Embodiments of the invention are constructed to minimize unwantedrefraction of emission and/or excitation signals that pass through thechip. As shown FIG. 1, the medium 52 that lies between the channel andthe collector comprises a solid, continuous medium such that lightpassing there between does not cross any boundaries where substantialrefraction can occur. As used herein, “substantial refraction” refers torefraction associated with a particular boundary that causes a decreasein the numerical aperture of an optical device by 5% or more. The“collector numerical aperture” for collectors is defined by a collectionray bundle that includes the detection zone, the margin of thecollector, and the detector. The “illuminator numerical aperture” forilluminators is defined by an illumination ray bundle that includes theemitter, the margin of the illuminator, and the detection zone.Similarly, the medium that lies between the illuminator and the channelcan be a solid, continuous medium. Such a solid, continuous medium canprevent substantial refraction of light passing there through. In oneembodiment of FIG. 1, the cover slip and the medium that lies betweenthe upper boundary of the channel, the collector and the illuminatorcomprises a monolithic piece of CORNING 550. CORNING 550 is favorable insome embodiments as it can be molded; however, other embodiments can usedifferent materials, as aspects of the invention are not limited to anyone type of material.

Optical components can be incorporated into the boundary of the solidmedium to further prevent unwanted refraction. For instance, theilluminator in the embodiment of FIG. 1 comprises a surface at theboundary 54 of the solid medium. It is shaped to refract an excitationsignal to the focal point at the channel. No further substantialrefraction occurs until the excitation signal exits the cover slip atthe channel boundary. The collector in the embodiment of FIG. 1comprises a coating of metal such as aluminum or silver with aprotective coating that is applied to the outer surface 56 of the mediumto maximize reflectance.

The illuminator and collector of the embodiment in FIG. 1 areincorporated into the chip by virtue of being part of the solid medium52 that includes the cover slip 58 of the chip. However, the opticalcomponents or other features can be incorporated into a chip indifferent manners. As used herein, the phrase “incorporated into a chip”refers to a component that is held to the chip in a way that causes thecomponent to move together with the chip. In this sense, when a chip ismoved or subjected to vibration, each component that is incorporatedinto the chip is subjected to the same movement or vibration. In thisregard, critical alignments between components that are incorporatedinto a chip are less susceptible to problems associated with chipmovement or chip/system vibrations.

As mentioned herein, relationships that are critical to satisfactoryoperation of a system, such as the relationship between an illuminatorand/or collector and a channel of a chip, can be made more robust byincorporating the optical components into the chip. For example, theposition of the detection zone relative to the channel (as defined byeither the illuminator or the collector) can be critical in determiningwhether an agent is present in the detection zone. If the detection zoneis moved to allow agents to pass through the channel without enteringthe detection zone, the system may register a false negative reading.Similarly, if the detection zone is positioned differently thanexpected, labels that are bound to polymers (e.g., via a probe) may beinterpreted to be at incorrect positions on the polymer, and thus leadto an incorrect interpretation of the polymer structure, includingpolymer sequence. As mentioned herein, incorporating the illuminatorand/or collector into the chip allows the detection zone to be held at afixed position relative to the channel, thus preventing problemsassociated with improper detection zone location.

The interface between the illuminator on the chip and the emitter in thesystem can be sensitive to angular alignment. Due to infinitely distantconjugates, the interface between the illuminator on the chip and theemitter in the system can be insensitive to spatial alignment.Therefore, the entire system may expand or contract without significantdegradation to the interface between the illuminator on the chip and anemitter as long as angular orientations are preserved. Duringmanufacture, the interface between the illuminator and the channel canbe sensitive to spatial alignment. During manufacture, the interfacebetween the illuminator on chip and the emitter can be sensitive toangular alignment. The emitter in the system can direct an excitationsignal that is larger in cross section than the exposed surface of theilluminator. In some embodiments, the illuminator will be nominallypositioned in the center of the laser so that if movement occurs, theilluminator may still be located entirely within the laser. The areassurrounding the illuminator on the chip can be opaque or reflective, sothat the excess excitation signal does not enter the chip. In someembodiments, the excitation signal comprises an area that is 50% greaterthan the cross sectional area of the illuminator. In other embodiments,the excitation signal comprises an area that is more than 100% greaterthan the cross sectional area of the illuminator, or even more than 200%times greater than the cross sectional area of the illuminator. In suchembodiments, the system can still operate even if the illuminator isonly partially bathed in the excitation signal. Here, the illuminatorwill still create a detection zone at the appropriate spot, but at alower intensity. In other embodiments where the excitation signal hasthe same or smaller cross sectional area as the illuminator, movement ofthe excitation signal about the surface of the illuminator will havelittle effect on the overall position of the detection zone but mayaffect the intensity at portions of the detection zone.

For reasons similar to those discussed herein with respect to theilluminator, the interface between the collector on the chip and thedetector in the system (or an intermediate optical component in thesystem, such as a mirror or filter) can be made robust to movement. Inmany embodiments, the surface of the detector or mirror that receivesthe detection signal directly from the chip has an area larger than thedetection signal that it is intended to receive. The larger area of thecollector or mirror can receive the detection signal even if there ismisalignment between the elements. This is yet another way in whichincorporating optical components into the chip can reduce a system'ssusceptibility to vibrations.

Components that are incorporated into the chip can be physicallyinseparable from the chip or a cover slip. For instance, in someembodiments one monolithic piece of material forms the chip, thecollector and the illuminator. In such embodiments, the collector andilluminator can be present on one side of the chip and the channel canbe exposed on the opposite or the opposed side of the chip that receivesa cover slip. In other embodiments, components are incorporated into achip, but can be separated there from. By way of example, in theembodiment of FIG. 1, the illuminator and collector are incorporatedinto the chip by virtue of being formed into the cover slip that mateswith the chip.

In still other embodiments, the components that are incorporated intothe chip can be separable from the chip or cover slip. FIG. 3 shows anexample of a separable cover slip, a separable collector and a separableilluminator that are incorporated into a chip. The chip includes amicrofluidic channel that receives and passes a sample that may containan agent through a detection zone in the channel. A cover slip is matedto the chip so that a lower surface of the slip provides an upperboundary 60 to the channel, thereby enclosing the channel. A concave,reflective collector is mated to the upper surface of the cover slip andsuch that, when assembled, the focal point of the collector is at thechannel. The collector includes a central aperture 40 in which aseparate, refractive illuminator is positioned. The illuminator is matedto the aperture in the collector such that the focal point of theilluminator is also positioned at the channel and coincident with thefocal point of the collector.

Separable components incorporated into a chip, like that of FIG. 3, canbe mated together in various ways. By way of example, the collectorshown in FIG. 3 includes a separate chip, cover slip, collector,illuminator and mirror that are incorporated into the chip. In someembodiments the optical components may need to be aligned precisely asthey are incorporated into the chip. For instance, the illuminator mayrequire close assembly tolerances for proper alignment of the detectionzone at the channel. To accommodate such assembly tolerances, in oneembodiment, the thickness of the illuminator is varied to improve thefocus of a collimated beam (i.e., the excitation signal) at the channel.Here, the illuminator thickness may be varied with an adhesive fillingthe gaps at the interface between the illuminator of the collector orthe illuminator may be ground to the optimum length for focus of theexcitation signal. In one embodiment, the lateral position of theilluminator is directed orthogonally onto the channel. During assembly,the lateral position of the illuminator can be actively aligned and thenfixed in place. Alignment may include positioning the illuminator tosatisfy an acceptable angular error between the emitter and theilluminator.

Some embodiments with separable optical components include registrationfeatures to aid in alignment during assembly. In one embodiment, thecollector receives the illuminator in only a single location thatpositions the illuminator and collector to have coincident focal points.In another embodiment, the chip, cover slip, and or collector can haverecessed portions adapted to receive and position mating components.Other components of the chip can have similar or different types oflocation aids, such as patterns on the chip, like a circle or squarethat mates with another component, as aspects of the present inventionare not limited in this regard. Registration features can be includedthat position any of the components relative to one another. By way ofnon-limiting example, posts may extend from the chip, through a coverslip and into a collector to directly register the collector withrespect to the chip. Still, other registration features are possible, asaspects of the invention are not limited in this regard.

In embodiments that include separable components that are incorporatedinto a chip, like the embodiment of FIG. 3, the interface betweenseparate optical components can be arranged to minimize the reflectionof light. This allows collection of light near and beyond the criticalangle for total internal reflection. By way of example, the interfacebetween the collector and the cover slip or the collector and theilluminator of FIG. 3 can include a thin layer of immersion oil toreduce the reflection of light crossing the interface. In otherembodiments, the interface includes a thin adhesive that holds thecomponents relative to one another and reduces the reflection of lightcrossing the interface.

Aspects of the present invention also facilitate collectors thatmaximize the amount of emission signals received by the collector. Thiscan prove particularly beneficial in systems that are used to detectsingle molecules. Collection efficiency is frequently quantified by anumerical aperture for a given lens, which is defined by Eq. 1 below andillustrated in FIG. 1.NA=n sin θ  Eq. 1

where:

-   -   NA=Numerical Aperture    -   n=Refractive index of the medium in which the collector operates    -   θ=Half angle between the collector and the channel

Numerical aperture can be maximized by increasing the half angle of thecollector. Incorporating the collector into the chip allows thecollector margin to be moved closer to the microfluidic channel. Acollector margin that lies closer to the channel can surround thechannel to a greater degree than a collector that is further away fromthe channel. In this manner, moving the collector margin closer to thechannel can increase the half angle of collection and thus the numericalaperture of the collector. Embodiments of the present invention havehalf angles up to 50 degrees, 75 degrees, 85 degrees and even up to 90degrees. A monolithic solid from the channel to the collector can have ahalf angle of 90 degrees. In other embodiments, numerical aperturesnormally achieved with a 90 degree half angle can be achieved by indexmatching of components that lie on the path between the channel and thereflector. In some embodiments, the reflector/collector has a numericalaperture of 1.0 or greater, 1.3 or greater, or 1.5 or greater. Thesenumerical apertures correspond to half angles of 39 degrees, 55 degrees,and 69 degrees, respectively within glass having a refractive index of1.60, such as CORNING 550. Half angles of 49 degrees, 78 degrees, and 90degrees will produce similar numerical apertures, respectively, inwater, which has an index of 1.33. It is to be understood that the halfangle is measured along the central axis of the collector although thecentral portion of collectors that include apertures do not effectivelycollect emissions. In this respect, Eq. 1 provides numerical aperturevalues, as recited in the specification or the claims, that are notcorrected for apertures within the collector. Under this currentdefinition of numerical aperture, the collection efficiency increasemonotonically with numerical aperture.

Illuminators and collectors can be manufactured specifically to theapplication for a given chip to reduce overall system costs. As is to beappreciated, compound lenses found in prior art systems are frequentlydesigned to accommodate a large spatial field and different wavelengthsof light. These requirements introduce complexities into the lens thatincrease cost. Incorporating optical components, such as collectors orilluminators, into a chip allows the components to be designed with onlya small field, thus reducing the number of lenses and other requiredcomponents. Similarly, the optical components incorporated into a chipmay only need to accommodate a narrow range of wavelengths (i.e., thewavelengths associated with excitation and emission signals that are tobe used in combination with the chip). Here, the illuminator can operateat essentially a single wavelength, thus reducing or eliminatingchromatic aberrations in the illuminator. The collector can operate in areflective mode with no chromatic aberrations due to reflection. Eachelement performs a specific task with a minimum complexity. Lower costmaterials may be available, such as some plastics, that can accommodatewavelengths of excitation and emission signals, while not necessarilyaccommodating others.AΩ=λ²  Eq. 2

The Etendue or space-angle product for Gaussian profile beam is definedby Eq. 2 above, in which A is the area of the spot, Ω is the solid angleof divergence, and λ is the wavelength. A Gaussian beam creates thesmallest space-angle possible. It can be preferred over the largerspace-angle products due to a circular or rectangular geometry. Theplanar version of the space-angle product can be defined below by Eq. 3in which d is the linear dimension of the spot, and β is the fullplane-angle of divergence. $\begin{matrix}{{d\quad\beta} = {\frac{4}{\pi}\lambda}} & {{Eq}.\quad 3}\end{matrix}$

The size of the illumination field is determined by the wavelength ofexcitation and the numerical aperture of the illuminator. For a circularspot, the diameter of the illumination field is defined by Eq. 4 below.$\begin{matrix}{d_{IF} = {{1.27\frac{\lambda}{2{NA}}} = {0.63\frac{\lambda}{n\quad\sin\quad\theta}}}} & {{Eq}.\quad 4}\end{matrix}$

An illumination field of 1.7 microns in width should have an NA 0.18 foran excitation wavelength of 488 nm. The half-angle within water shouldbe 7.8 degrees. The half-angle within glass of index 1.5 should be 13.5degrees. The depth of focus is determined by a doubling of the spot areaat which point the peak irradiance is cut in half and the diameter isscaled by 1.4. The depth of focus in channel for circular spot isdefined by Eq. 5 below, where λ_(C) is the wavelength in the channel,Ω_(C) is the solid angle in the channel, n_(C) is the refractive indexof the channel, λ₀ is the wavelength within air. $\begin{matrix}{{\Delta\quad z_{IF}} = {\frac{\lambda_{C}}{\Omega_{C}} \approx \frac{n_{C}\lambda_{0}}{\pi\quad{NA}^{2}}}} & {{Eq}.\quad 5}\end{matrix}$

For a circular spot within water, the depth of focus is ±05.8 um. Anelliptical spot may be created by an elliptical beam at the illuminatoraperture. Its width and depth of focus are the same as the circularspot. However, over the same depth of focus for a circular spot, thepeak intensity is not cut in half. The peak intensity of the ellipticalspot is scaled by 0.71 over the depth of focus. Therefore and ellipticalbeam at the aperture of the illuminator may be advantageous due to asmaller drop in irradiance throughout the depth of focus.

The size and shape of the detection zone can be tailored by the designof the illuminator and/or reflector. In many embodiments, particularlythose suited for single molecule detection, it is desirable for thedetection zone to extend entirely across the channel in a directionperpendicular to flow. This can prevent an agent from passing by thedetection zone without being excited by the excitation signal. It canalso be desirable to minimize the length of the detection zone in adirection parallel to flow in the channel. Many systems cannot determinethe precise location of an agent within a detection zone, but rather cansimply detect whether the agent is present. In this regard, detectionzones that extend further in a direction parallel to flow can produceresults with less precision. To this end, it can be advantageous to havean excitation zone that extends a relatively short distance in thedirection parallel to flow, and in some embodiments as short as about1.7 microns or much shorter. Optical components can be constructed tocreate detection zones that extend across and beyond a channel in adirection perpendicular to flow and that extend a shorter distance in adirection parallel to flow. By way of example, an illuminator or theexcitation signal can be shaped to produce an elliptical detection zonewith a major axis extending across a channel and a minor axis extendingalong the channel. In one embodiment, the major axis is approximately 25microns and the minor axis is approximately 1.7 microns. The effectivefocal length can be 2.5 mm, therefore the angular tolerance the beam 10mrad or 0.5 degrees. In another embodiment, the detection zone iscircular with a diameter of approximately 1.7 microns, (whereapproximately indicates +/−0.2 microns, when used with reference to thedimensions of a detection zone). However, it is to be appreciated thatother shapes and sizes of detection zones are possible, as aspects ofthe present invention are not limited in this respect.

Optical components can be arranged in various ways about the chip toaccomplish effects similar to those of the embodiment shown in FIG. 1.As shown in FIG. 4, the illuminator can be positioned on a differentside of the channel than the collector. Here, the excitation signal isreceived by a refractive illuminator that focuses the excitation signalat the channel to create a detection zone. The concave, reflectivecollector has a focal point at the detection zone to receive a detectionsignal that will include emission signals from any agents present in thedetection zone. As in the embodiment of FIG. 1, the concave reflectorincludes a central aperture through which the excitation signal passesto prevent the excitation signal from being reflected as a component ofthe detection signal. Due to the central aperture in the collector, thecollector reflects an annulus of light toward the illuminator andultimately toward a detector in the system. The excitation signal canpass through the center of the annulus to prevent the overlap of theexcitation signal with the emission signals, as in the embodiment ofFIG. 1.

Optical elements, such as a mirror or filters, can be used inembodiments of the invention to modify excitation, detection, and/oremission signals. By way of example, optical elements can includedichroic mirrors tuned to allow passage of light with wavelengths atthat of the excitation signal, while reflecting light at wavelengthsassociated with expected emission signals. In one embodiment, theexcitation signal passes through the dichroic mirror both when travelingfrom the illuminator to the detection zone and from the detection zoneback toward the illuminator. However, spectral portions of the detectionsignal, that may include emission signals, are reflected by the dichroicmirror downstream toward a detector while portions of the detectionsignal at wavelengths associated with the excitation signal are notdirected toward the detector.

In one embodiment, a mirror can include a central aperture positioned toallow passage of a portion of the detection signal that includes theexcitation signal, while the surrounding mirror reflects spatialportions of the detection signal that do not include the excitationsignal. In such an embodiment, excitation signals that have beenoverlapped with the emission signal are removed from the detectionsignal prior to reaching the detector.

The chip can alternately incorporate a reflective illuminator and acollector on opposed sides of a channel, as shown in FIG. 5. Thereflective illuminator directs an excitation signal received from thesystem towards the channel in the chip to define a detection zone. Aconcave, reflective collector receives a detection signal from thedetection zone, including any emission signals from agents therein, andreflects the detection signal toward a downstream detector. In theembodiment illustrated in FIG. 5, the reflective collector includes anaperture positioned to allow passage of the excitation signal, such thatthe excitation signal is not reflected toward the downstream detector asa component of the detection signal. In alternate embodiments, theexcitation signal can be removed from the detection signal by a filter,such as a dichroic mirror, or by a mirror that selectively reflectsspatial portions of the detection signal that do not include theexcitation signal, as described herein.

Chips can include a plurality of paired illuminators and collectors,along with other optical components, to increase the amount ofinformation that can be obtained from a sample during an assay. In someembodiments, paired illuminators and collectors are positioned along thelength of a common channel so that a sample passes through detectionzones associated with each of the illuminators in a serial manner, likethat illustrated in FIG. 6. In other embodiments, a chip includesmultiple, separate channels each having one more pairs of collectors andilluminators. In some embodiments, such serially arranged detectionzones can be used to produce redundant information about an agent, asdescribed in U.S. Provisional Patent Application Ser. No. 60/630,902filed on Nov. 24, 2004, entitled LINEAR ANALYSIS OF POLYMERS, which ishereby incorporated by reference in its entirety. In some embodiments,serially arranged detection zones can be associated with differentexcitation signals, each tuned for the detection and/or analysis of aparticular agent or label thereon. In this manner, multiple, differentassays can be performed for different agents (or labels0 in a sample asthe sample passes through the chip. Chips can also include multiplechannels arranged in parallel to decrease sample throughput time in achip, as shown in FIG. 7. Arranging channels in parallel allows moresample to be processed in an equivalent amount of time, all elseconstant. The channels can be fluidly connected to one another to allowa common sample to pass through each of the channels. In otherembodiments, the chip includes multiple channels that are fluidlyisolated from one another. Each of the isolated channels may have itsown supply and exit port on the chip.

Samples may be passed through microfluidic devices in the system or thechip prior to being passed through a detection zone. Such microfluidicdevices can be used to linearize or stretch an agent, like a polymer,prior to analysis. However, this may not be necessary if the ultimatedetection system is capable of analyzing both stretched and condensedpolymers. As used herein, stretching of the polymer means that thepolymer is provided in a substantially linear extended form rather thana compacted, coiled and/or folded form. Stretching the polymer prior toanalysis may be accomplished using any of the microfluidic devices ortechniques discussed in U.S. patent application Ser. No. 10/821,664,filed on Apr. 9, 2004, entitled ADVANCED MICROFLUIDICS and publishedunder publication no. US-2005-0112606-A1. These configurations may notbe required if the target polymer can be analyzed in a compacted form.

Illuminators can also be incorporated into the chips of someembodiments. In some embodiments, a waveguide is embedded into the chip.The waveguide can pass adjacent to a channel in the chip to provide anevanescent excitation signal to the channel to define a detection zone.In some embodiments, a single waveguide can pass adjacent multiplechannels, or multiple portions of a common channel to create multipledetection zones. In other embodiments, the waveguide terminates at thesurface of the channel where it creates a detection zone. In suchembodiments, the waveguide tapers as it approaches to create anevanescent excitation signal at the channel.

FIG. 8 displays waveguide with evanescent field extending into thefluidic channel, according to one embodiment. The waveguide comprises awaveguide channel incorporated into the chip. The refractive index ofthe waveguide is higher than the refractive index of the chip. Theevanescent field 25 of the waveguide extends through the depth of thefluidic channel. The evanescent field decays exponentially as a functionof distance from the waveguide, typically within 200 nanometers forvisible wavelengths. Therefore, in some embodiments, with channels thatare deeper than 200 nanometers, only a portion the channel depth isilluminated. The waveguide channel may directly contact fluidic channelor the waveguide channel may be separated from the fluidic channel by adielectric film. The waveguide channel may cross a plurality of fluidicchannels. Embodiments that utilize a waveguide incorporated into thechip as an illuminator do not require a clear aperture to separateexcitation signals from the detection signal, due to the evanescentnature of the excitation signal.

Samples can be derived from virtually any source known to contain orsuspected of containing an agent of interest. Samples can be of solid,liquid or gaseous nature. They may be purified but usually are not.Different samples can be collected from different environments andprepared in the same manner by using the appropriate collecting device.

The samples to be tested can be a biological or bodily sample such as atissue biopsy, urine, sputum, semen, stool, saliva and the like. Theinvention further contemplates preparation and analysis of samples thatmay be biowarfare targets. Air, liquids and solids that will come intocontact with the greatest number of people are most likely to bebiowarfare targets. Samples to be tested for the presence of such agentsmay be taken from an indoor or outdoor environment. Such biowarfaresampling can occur continuously, although this may not be necessary inevery application. For example, in an airport setting, it may only benecessary to harvest randomly a sample near or around select baggage. Inother instances, it may be necessary to continually monitor (and thussample) the environment. These instances may occur in “heightened alert”states. In some important embodiments, the sample is tested for thepresence of a pathogen. Samples can be tested for the presence ofpathogenic substances such as but not limited to food pathogens,water-borne pathogens, and aerosolized pathogens.

Liquid samples can be taken from public water supplies, waterreservoirs, lakes, rivers, wells, springs, and commercially availablebeverages. Solids such as food (including baby food and formula), money(including paper and coin currencies), public transportation tokens,books, and the like can also be sampled via swipe, wipe or swab testingand by placing the swipe, wipe or swab in a liquid for dissolution ofany agents attached thereto. Based on the size of the swipe or swab andthe volume of the corresponding liquid it must be placed in for agentdissolution, it may or may not be necessary to concentrate such liquidsample prior to further manipulation.

Air samples can be tested for the presence of normally airbornesubstances as well as aerosolized (or weaponized) chemicals or biologicsthat are not normally airborne. Air samples can be taken from a varietyof places suspected of being biowarfare targets including public placessuch as airports, hotels, office buildings, government facilities, andpublic transportation vehicles such as buses, trains, airplanes, and thelike.

The choice of air sampling instruments is dependent on userrequirements, and those of ordinary skill in the art will be able toidentify the appropriate instrument for a particular application.Various air sampling devices are currently commercially available, fromcompanies such as BioAerosol Concentrator, International pbi S.pA., MesoSystems, Sceptor Industries, Inc., and Anderson. Moreover, techniquesfor air sampling are described in J. P. Lodge, Jr. Methods of AirSampling and Analysis, Third Edition, Lewis Publishers, Inc. (Dec. 31,1988) ISBN 0873711416.

The agent is any molecule to be detected using the systems and methodsprovided herein. It may be a biological or chemical in nature, but isnot so limited. It may be naturally or non-naturally occurring,including agents synthesized ex vivo but released into a naturalenvironment. Agents include but are not limited to proteins, nucleicacids, chemicals and the like. The agents may be biohazardous agents asdescribed in greater detail herein. As described herein, the methods andsystems of the invention can be used to detect one or more agentsconcurrently, simultaneously or consecutively. A plurality of agents ismore than one and less than an infinite number. It includes less than10¹⁰, less than 10⁹, less than 10⁸, less than 10⁷, less than 10⁶, lessthan 10⁵, less than 10⁴, less than 5000, less than 1000, less than 500,less 100, less than 50, less than 25, less than 10 and less than 5, aswell as every integer therebetween as if explicitly recited herein.

The invention can be applied to the detection and optionallyidentification and/or quantification of any agent, but most preferablyrare agents which would otherwise be costly to detect. One example ofsuch agents is biohazardous or biowarfare agents. These agents can bebiological or chemical in nature. Biological biowarfare agents can beclassified broadly as pathogens (including spores thereof) or toxins. Asused herein, a pathogen (including a spore thereof) is an agent capableof entering a subject such as a human and infecting that subject.Examples of pathogens include infectious agents such bacteria, viruses,fungi, parasites, mycobacteria and the like. Prions may also beconsidered pathogens to the extent they are thought to be thetransmitting agent for CJD and like diseases. As used herein, a toxin isa pathogen-derived agent that causes disease and often death in asubject without also causing an infection. It derives from pathogens andso may be harvested therefrom. Alternatively, it may be synthesizedapart from pathogen sources. Biologicals may be weaponized (i.e.,aerosolized) for maximum spread.

In some embodiments, the agents are detected directly via the use ofprobes that bind to the agent itself. In other embodiments, the agentsare detected via the use of probes that bind to agent specificcomponents such as agent specific nucleic acids (e.g., DNA).

CDC Category A agents include Bacillus anthracis (otherwise known asanthrax), Clostridium botulinum and its toxin (causative agent forbotulism), Yersinia pestis (causative agent for the plague), variolamajor (causative agent for small pox), Francisella tularensis (causativeagent for tularemia), and viral hemorrhagic fever causing agents such asfiloviruses Ebola and Marburg and arenaviruses such as Lassa, Machupoand Junin.

CDC Category B agents include Brucellosis (Brucella species), epsilontoxin of Clostridium perfringens, food safety threats such as Salmonellaspecies, E. coli and Shigella, Glanders (Burkholderia mallei),Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydiapsittaci), Q fever (Coxiella burnetii), ricin toxin (from Ricinuscommunis—castor beans), Staphylococcal enterotoxin B, Typhus fever(Rickettsia prowazekii), viral encephalitis (alphaviruses, e.g.,Venezuelan equine encephalitis, eastern equine encephalitis, westernequine encephalitis), and water safety threats such as e.g., Vibriocholerae, Cryptosporidium parvum.

CDC Category C agents include emerging infectious diseases such as Nipahvirus and hantavirus.

Further examples of bacteria that can be used as biohazards includeGonorrhea, Staphylococcus spp., Streptococcus spp. such as Streptococcuspneumoniae, Syphilis, Pseudomonas spp., Clostridium difficile,Legionella spp., Pneumococcus spp., Haemophilus spp. (e.g., Haemophilusinfluenzae), Klebsiella spp., Enterobacter spp., Citrobacter spp.,Neisseria spp. (e.g., N. meningitidis, N. gonorrhoeae), Shigella spp.,Salmonella spp., Listeria spp. (e.g., L. monocytogenes), Pasteurellaspp. (e.g., Pasteurella multocida), Streptobacillus spp., Spirillumspp., Treponema spp. (e.g., Treponema pallidum), Actinomyces spp. (e.g.,Actinomyces israelli), Borrelia spp., Corynebacterium spp., Nocardiaspp., Gardnerella spp. (e.g., Gardnerella vaginalis), Campylobacterspp., Spirochaeta spp., Proteus spp., and Bacteriodes spp.

Further examples of viruses that can be used as biohazards includeHepatitis virus A, B and C, West Nile virus, poliovirus, rhinovirus,HIV, Herpes simplex virus 1 and 2 (including encephalitis, neonatal andgenital forms), human papilloma virus, cytomegalovirus, Epstein Barrvirus, Hepatitis virus A, B and C, rotavirus, adenovirus, influenzavirus including influenza A virus, respiratory syncytial virus,varicella-zoster virus, small pox, monkey pox and SARS virus.

Further examples of fungi that can be used as biohazards includecandidiasis, ringworm, histoplasmosis, blastomycosis,paracoccidioidomycosis, crytococcosis, aspergillosis, chromomycosis,mycetoma, pseudallescheriasis, and tinea versicolor.

Further examples of parasites that can be used as biohazards includeboth protozoa and nematodes such as amebiasis, Trypanosoma cruzi,Fascioliasis (e.g., Facioloa hepatica), Leishmaniasis, Plasmodium (e.g.,P. falciparum, P. knowlesi, P. malariae), Onchocerciasis,Paragonimiasis, Trypanosoma brucei, Pneumocystis (e.g., Pneumocystiscarinii), Trichomonas vaginalis, Taenia, Hymenolepsis (e.g.,Hymenolepsis nana), Echinococcus, Schistosomiasis (e.g., Schistosomamansoni), neurocysticercosis, Necator americanus, and Trichuristrichuria, Giardia.

Further examples of mycobacteria that can be used as biohazards includeM. tuberculosis or M. leprae.

Examples of toxins include abrin, ricin and strychnine. Further examplesof toxins include toxins produced by Corynebacterium diphtheriae(diphtheria), Bordetella pertussis (whooping cough), Vibrio cholerae(cholera), Bacillus anthracis (anthrax), Clostridium botulinum(botulism), Clostridium tetani (tetanus), and enterohemorrhagicEscherichia coli (bloody diarrhea and hemolytic uremic syndrome),Staphylococcus aureus alpha toxin, Shiga toxin (ST), cytotoxicnecrotizing factor type 1 (CNF1), E. coli heat-stable toxin (ST),botulinum, tetanus neurotoxins, S. aureus toxic shock syndrome toxin(TSST), Aeromonas hydrophila aerolysin, Clostridium perfringensperfringolysin O, E. coli hemolysin, Listeria monocytogeneslisteriolysin O, Streptococcus pneumoniae pneumolysin, Streptococcuspyogenes streptolysine O, Pseudomonas aeruginosa exotoxin A, E. coliDNF, E. coli LT, E. coli CLDT, E. coli EAST, Bacillus anthracis edemafactor, Bordetella pertussis dermonecrotic toxin, Clostridium botulinumC2 toxin, C. botulinum C3 toxin, Clostridium difficile toxin A, and C.difficile toxin B.

Examples of chemicals that can be detected include arsenic, arsine,benzene, blister agents/vesicants, blood agents, bromine,borombenzylcyanide, chlorine, choking/lung/pulmonary agents, cyanide,distilled mustard, fentanyls and other opioids, mercury, mustard gas,nerve agents, nitrogen mustard, organic solvents, paraquat, phosgene,phosphine, sarin, sesqui mustard, stibine, sulfur mustard, warfarin,tabun, and the like.

The foregoing lists of infections are not intended to be exhaustive butrather exemplary.

It may be necessary to disrupt pathogen cell walls, cell membranes orviral envelopes, in some embodiments. This can enrich for agents to bedetected. Disruption can be accomplished by any number of meansincluding mechanical, electrical, osmotic, pressure, and the like. Inone embodiment, the sample is exposed to an acoustic conditioningmethod.

As an example, microorganisms can be disrupted using a non-contact,reagent-less focused acoustic technology, developed at Covaris Inc.,Woburn, Mass., and described in U.S. Pat. No. 6,719,449, issued Apr. 13,2004. This procedure enables higher recoveries and betterreproducibility than conventional, physical contact systems such asliquid nitrogen grinding, bead beating, sonicators (low frequency,unfocused, standing waves) and polytron-type homogenizers.

The agents may be processed using one or more reagents that acts on orreacts with and thereby modifies the agent. The nature of the reagentswill vary depending on the processing step being performed with suchreagent. The reagent may be a lysing agent (e.g., a detergent such asbut not limited to deoxycholate), a labeling agent or probe (e.g., asequence-specific nucleic acid probe), an enzyme (e.g., a nuclease suchas a restriction endonuclease), an enzyme co-factor, a stabilizer (e.g.,an anti-oxidant), and the like. One of ordinary skill in the art canenvision other reagents to be used.

By way of example, a fluid may contain a lysing agent that lysescellular agents (e.g., mammalian cells or pathogens such as bacteria,viruses and the like) in the channel, thereby releasing cellularcontents, such as nucleic acids, into the channel. The fluids used inthe invention may contain other components such as buffering compounds(e.g., TRIS), chelating compounds (e.g., EDTA), ions (e.g., monovalent,divalent or trivalent cations or anions), salts, and the like.

The agent may be a polymer. A “polymer” as used herein is a compoundhaving a linear backbone to which monomers are linked together bylinkages. The polymer is made up of a plurality of individual monomers.An individual monomer as used herein is the smallest building block thatcan be linked directly or indirectly to other building blocks (ormonomers) to form a polymer. At a minimum, the polymer contains at leasttwo linked monomers. The particular type of monomer will depend upon thetype of polymer being analyzed. The polymer may be a nucleic acid, apeptide including a protein, a carbohydrate, an oligo- orpolysaccharide, a lipid, etc.

In some embodiments, the polymer is capable of being bound to or bysequence- or structure-specific probes, wherein the sequence orstructure recognized and bound by the probe is unique to that polymer orto a region of the polymer. It is possible to use a given probe for twoor more polymers if a polymer is recognized by two or more probes,provided that the combination of probes is still specific for only agiven polymer. The sample in some instances can be analyzed as iswithout harvest and isolation of polymers contained therein.

In some embodiments, the method can be used to detect a plurality ofdifferent polymers in a sample.

In some important embodiments, the agents are polymers such as nucleicacids. The term “nucleic acid” refers to multiple linked nucleotides(i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linkedto an exchangeable organic base, which is either a pyrimidine (e.g.,cytosine (C), thymidine (T) or uracil (U)) or a purine (e.g., adenine(A) or guanine (G)). “Nucleic acid” and “nucleic acid molecule” are usedinterchangeably and refer to oligoribonucleotides as well asoligodeoxyribonucleotides. The terms shall also include polynucleosides(i.e., a polynucleotide minus a phosphate) and any other organic basecontaining nucleic acid. The organic bases include adenine, uracil,guanine, thymine, cytosine and inosine.

In important embodiments, the nucleic acid is Deoxyribonucleic Acid(DNA) or Ribonucleic Acid (RNA). DNA includes genomic DNA (such asnuclear DNA and mitochondrial DNA), as well as in some instancescomplementary DNA (cDNA). RNA includes messenger RNA (mRNA), miRNA,siRNA and the like. Non-naturally occurring nucleic acids include butare not limited to bacterial artificial chromosomes (BACs) and yeastartificial chromosomes (YACs). Harvest and isolation of nucleic acidsare routinely performed in the art and suitable methods can be found instandard molecular biology textbooks. (See, for example, Maniatis'Handbook of Molecular Biology.)

Preferably, prior amplification using techniques such as polymerasechain reaction (PCR) are not necessary. Accordingly, the polymer may bea non in vitro amplified nucleic acid. As used herein, a “non in vitroamplified nucleic acid” refers to a nucleic acid that has not beenamplified in vitro using techniques such as polymerase chain reaction orrecombinant DNA methods. A non in vitro amplified nucleic acid mayhowever be a nucleic acid that is amplified in vivo (in the biologicalsample from which it was harvested) as a natural consequence of thedevelopment of the cells in vivo. This means that the non in vitronucleic acid may be one which is amplified in vivo as part of locusamplification, which is commonly observed in some cell types as a resultof mutation or cancer development.

As used herein with respect to linked units of a polymer including anucleic acid, “linked” or “linkage” means two entities bound to oneanother by any physicochemical means. Any linkage known to those ofordinary skill in the art, covalent or non-covalent, is embraced.Natural linkages, which are those ordinarily found in nature connectingfor example the individual units of a particular nucleic acid, are mostcommon. Natural linkages include, for instance, amide, ester andthioester linkages. The individual units of a nucleic acid analyzed bythe methods of the invention may be linked, however, by synthetic ormodified linkages. Nucleic acids where the units are linked by covalentbonds will be most common but those that include hydrogen bonded unitsare also embraced by the invention. It is to be understood that allpossibilities regarding nucleic acids apply equally to nucleic acidtargets and nucleic acid probes, as discussed herein.

The nucleic acids may be double-stranded, although in some embodimentsthe nucleic acid targets are denatured and presented in asingle-stranded form. This can be accomplished by modulating theenvironment of a double-stranded nucleic acid including singly or incombination increasing temperature, decreasing salt concentration, andthe like. Methods of denaturing nucleic acids are known in the art.

The target nucleic acids commonly have a phosphodiester backbone becausethis backbone is most common in vivo. However, they are not so limited.Backbone modifications are known in the art. One of ordinary skill inthe art is capable of preparing such nucleic acids without undueexperimentation. The probes, if nucleic acid in nature, can also havebackbone modifications such as those described herein.

Thus the nucleic acids may be heterogeneous in backbone compositionthereby containing any possible combination of nucleic acid units linkedtogether such as peptide nucleic acids (which have amino acid linkageswith nucleic acid bases, and which are discussed in greater detailherein). In some embodiments, the nucleic acids are homogeneous inbackbone composition.

The methods of the invention in part may be used to analyze agents usingprobes that recognize and specifically bind to an agent. Binding of aprobe to an agent may indicate the presence and location of a targetsite in the target agent, or it may simply indicate the presence of theagent, depending on user requirements. As used herein, a target agentthat is bound by a probe is “labeled” with the probe and/or itsdetectable label.

As used herein, a probe is a molecule or compound that bindspreferentially to the agent of interest (i.e., it has a greater affinityfor the agent of interest than for other compounds). Its affinity forthe agent of interest may be at least 2-fold, at least 5-fold, at least10-fold, or more than its affinity for another compound. Probes with thegreatest differential affinity are preferred in most embodiments.

The probes can be of any nature including but not limited to nucleicacid (e.g., aptamers), peptide, carbohydrate, lipid, and the like. Anucleic acid based probe such as an oligonucleotide can be used torecognize and bind DNA or RNA. The nucleic acid based probe can be DNA,RNA, Locked Nucleic Acid (LNA) or Peptide Nucleic Acid (PNA), althoughit is not so limited. It can also be a combination of one or more ofthese elements and/or can comprise other nucleic acid mimics. With theadvent of aptamer technology, it is possible to use nucleic acid basedprobes in order to recognize and bind a variety of compounds, includingpeptides and carbohydrates, in a structurally, and thus sequence,specific manner. Other probes for nucleic acid targets include but arenot limited to sequence-specific major and minor groove binders andintercalators, nucleic acid binding peptides or proteins, etc.

As used herein a “peptide” is a polymer of amino acids connectedpreferably but not solely with peptide bonds. The probe may be anantibody or an antibody fragment. Antibodies include IgG, IgA, IgM, IgE,IgD as well as antibody variants such as single chain antibodies.Antibody fragments contain an antigen-binding site and thus include butare not limited to Fab and F(ab)₂ fragments.

The methods provided herein involve the use of probes that bind to thetarget polymer in a sequence-specific manner. “Sequence-specific” whenused in the context of a nucleic acid means that the probe recognizes aparticular linear (or in some instances quasi-linear) arrangement ofnucleotides or derivatives thereof. In some embodiments, the probes are“polymer-specific” meaning that they bind specifically to a particularpolymer, possibly by virtue of a particular sequence or structure uniqueto that polymer.

In some instances, nucleic acid probes will form at least a Watson-Crickbond with a target nucleic acid. In other instances, the nucleic acidprobe can form a Hoogsteen bond with the target nucleic acid, therebyforming a triplex. Examples of these latter probes include moleculesthat recognize and bind to the minor and major grooves of nucleic acids(e.g., some forms of antibiotics). In some embodiments, the nucleic acidprobes can form both Watson-Crick and Hoogsteen bonds with the nucleicacid polymer. Bis PNA probes, for instance, are capable of bothWatson-Crick and Hoogsteen binding to a nucleic acid.

The nucleic acid probes of the invention can be any length ranging fromat least 4 nucleotides to in excess of 1000 nucleotides. The length ofthe probe can be any length of nucleotides between and including theranges listed herein, as if each and every length was explicitly recitedherein.

The probes are preferably single-stranded, but they are not so limited.

The nucleic acid probe hybridizes to a complementary sequence within thenucleic acid polymer. The specificity of binding can be manipulatedbased on the hybridization conditions. For example, salt concentrationand temperature can be modulated in order to vary the range of sequencesrecognized by the nucleic acid probes. Those of ordinary skill in theart will be able to determine optimum conditions for a desiredspecificity.

In some embodiments, the probes may be molecular beacons. When not boundto their targets, the molecular beacon probes form a hairpin structureand do not emit fluorescence since one end of the molecular beacon is aquencher molecule. However, when bound to their targets, the fluorescentand quenching ends of the probe are sufficiently separated so that thefluorescent end can now emit.

The probes may be nucleic acids, as described herein, or nucleic acidderivatives. As used herein, a “nucleic acid derivative” is anon-naturally occurring nucleic acid or a unit thereof. Nucleic acidderivatives may contain non-naturally occurring elements such asnon-naturally occurring nucleotides and non-naturally occurring backbonelinkages. These include substituted purines and pyrimidines such as C-5propyne modified bases, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouraciland pseudoisocytosine. Other such modifications are well known to thoseof skill in the art.

The nucleic acid derivatives may also encompass substitutions ormodifications, such as in the bases and/or sugars. For example, theyinclude nucleic acids having backbone sugars which are covalentlyattached to low molecular weight organic groups other than a hydroxylgroup at the 3′ position and other than a phosphate group at the 5′position. Thus, modified nucleic acids may include a 2′-O-alkylatedribose group. In addition, modified nucleic acids may include sugarssuch as arabinose instead of ribose.

In some embodiments, the probe is a nucleic acid that is a PNA, a bisPNA clamp, a pseudocomplementary PNA, a locked nucleic acid (LNA), DNA,RNA, or co-nucleic acids of the above such as DNA-LNA co-nucleic acids.siRNA or miRNA or RNAi molecules can be similarly used.

In some embodiments, the probe is a peptide nucleic acid (PNA), a bisPNA clamp, a locked nucleic acid (LNA), a ssPNA, a pseudocomplementaryPNA (pcPNA), a two-armed PNA (as described in co-pending U.S. patentapplication having Ser. No. 10/421,644 and publication number US2003-0215864 A1 and published Nov. 20, 2003, and PCT application havingserial number PCT/US03/12480 and publication number WO 03/091455 A1 andpublished Nov. 6, 2003, filed on Apr. 23, 2003), or co-polymers thereof(e.g., a DNA-LNA co-polymer).

As stated herein, the agent may be labeled either by binding a probe. Asan example, if the agent is a nucleic acid, it may be labeled throughthe use of sequence-specific probes that bind to the polymer in asequence-specific manner. The sequence-specific probes are labeled witha detectable label. The nucleic acid however can also be synthesized ina manner that incorporates detectable labels directly into the growingnucleic acid. For example, this latter labeling can be accomplished bychemical means or by the introduction of active amino or thiol groupsinto nucleic acids. (Proudnikov and Mirabekov, Nucleic Acid Research,24:4535-4532, 1996.) An extensive description of modification proceduresthat can be performed on a nucleic acid polymer can be found inHermanson, G. T., Bioconjugate Techniques, Academic Press, Inc., SanDiego, 1996, which is incorporated by reference herein.

There are several known methods of direct chemical labeling of DNA(Hermanson, 1996; Roget et al., 1989; Proudnikov and Mirabekov, 1996).One of the methods is based on the introduction of aldehyde groups bypartial depurination of DNA. Fluorescent labels with an attachedhydrazine group are efficiently coupled with the aldehyde groups and thehydrazine bonds are stabilized by reduction with sodium labelingefficiencies around 60%. The reaction of cytosine with bisulfite in thepresence of an excess of an amine fluorophore leads to transamination atthe N4 position (Hermanson, 1996). Reaction conditions such as pH, aminefluorophore concentration, and incubation time and temperature affectthe yield of products formed. At high concentrations of the aminefluorophore (3M), transamination can approach 100% (Draper and Gold,1980).

In addition to the above method, it is also possible to synthesizenucleic acids de novo (e.g., using automated nucleic acid synthesizers)using fluorescently labeled nucleotides. Such nucleotides arecommercially available from suppliers such as Amersham PharmaciaBiotech, Molecular Probes, and New England Nuclear/Perkin Elmer.

Probes are generally labeled with a detectable label. A detectable labelis a moiety, the presence of which can be ascertained directly orindirectly. Generally, detection of the label involves the creation of adetectable signal such as for example an emission of energy. The labelmay be of a chemical, peptide or nucleic acid nature although it is notso limited. The nature of label used will depend on a variety offactors, including the nature of the analysis being conducted, the typeof the energy source and detector used and the type of polymer andprobe. The label should be sterically and chemically compatible with theconstituents to which it is bound.

The label can be detected directly for example by its ability to emitand/or absorb electromagnetic radiation of a particular wavelength. Alabel can be detected indirectly for example by its ability to bind,recruit and, in some cases, cleave another moiety which itself may emitor absorb light of a particular wavelength (e.g., an epitope tag such asthe FLAG epitope, an enzyme tag such as horseradish peroxidase, etc.).Generally the detectable label can be selected from the group consistingof directly detectable labels such as a fluorescent molecule (e.g.,fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3,Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), fluoresceinamine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), 6carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS), 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid,acridine, acridine isothiocyanate,r-amino-N-(3-vinylsulfonyl)phenylnaphthalimide-3,5, disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin, 7-amino-4-methylcoumarin,7-amino-4-trifluoromethylcouluarin (Coumarin 151), cyanosine,4′,6-diaminidino-2-phenylindole (DAPI), 5′,5″-diaminidino-2-phenylindole(DAPI), 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosinisothiocyanate, erythrosin B, erythrosin isothiocyanate, ethidium,5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), QFITC (XRITC),fluorescamine, IR144, IR1446, Malachite Green isothiocyanate,4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene,pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4(Cibacron. RTM. Brilliant Red 3B-A), lissamine rhodamine B sulfonylchloride, rhodamine B, rhodamine 123, rhodamine X, sulforhodamine B,sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101,tetramethyl rhodamine, riboflavin, rosolic acid, and terbium chelatederivatives), a chemiluminescent molecule, a bioluminescent molecule, achromogenic molecule, a radioisotope (e.g., P³² or H³, ¹⁴C, ¹²⁵I and¹³¹I), an electron spin resonance molecule (such as for example nitroxylradicals), an optical or electron density molecule, an electrical chargetransducing or transferring molecule, an electromagnetic molecule suchas a magnetic or paramagnetic bead or particle, a semiconductornanocrystal or nanoparticle (such as quantum dots described for examplein U.S. Pat. No. 6,207,392 and commercially available from Quantum DotCorporation and Evident Technologies), a colloidal metal, a colloid goldnanocrystal, a nuclear magnetic resonance molecule, and the like.

The detectable label can also be selected from the group consisting ofindirectly detectable labels such as an enzyme (e.g., alkalinephosphatase, horseradish peroxidase, β-galactosidase, glucoamylase,lysozyme, luciferases such as firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456); saccharide oxidases such asglucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase; heterocyclic oxidases such as uricase and xanthineoxidase coupled to an enzyme that uses hydrogen peroxide to oxidize adye precursor such as HRP, lactoperoxidase, or microperoxidase), anenzyme substrate, an affinity molecule, a ligand, a receptor, a biotinmolecule, an avidin molecule, a streptavidin molecule, an antigen (e.g.,epitope tags such as the FLAG or HA epitope), a hapten (e.g., biotin,pyridoxal, digoxigenin fluorescein and dinitrophenol), an antibody, anantibody fragment, a microbead, and the like. Antibody fragments includeFab, F(ab)₂, Fd and antibody fragments which include a CDR3 region.

In some embodiments, the detectable label is a member of a FRETfluorophore pair. FRET fluorophore pairs are two fluorophores that arecapable of undergoing FRET to produce or eliminate a detectable signalwhen positioned in proximity to one another. Examples of donors includeAlexa 488, Alexa 546, BODIPY 493, Oyster 556, Fluor (FAM), Cy3 and TMR(Tamra). Examples of acceptors include Cy5, Alexa 594, Alexa 647 andOyster 656. Cy5 can work as a donor with Cy3, TMR or Alexa 546, as anexample. FRET should be possible with any fluorophore pair havingfluorescence maxima spaced at 50-100 nm from each other.

The polymer may be labeled in a sequence non-specific manner. Forexample, if the polymer is a nucleic acid such as DNA, then its backbonemay be stained with a backbone label. Examples of backbone stains thatlabel nucleic acids in a sequence non-specific manner includeintercalating dyes such as phenanthridines and acridines (e.g., ethidiumbromide, propidium iodide, hexidium iodide, dihydroethidium, ethidiumhomodimer-1 and -2, ethidium monoazide, and ACMA); minor grove binderssuch as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342,Hoechst 34580 and DAPI); and miscellaneous nucleic acid stains such asacridine orange (also capable of intercalating), 7-AAD, actinomycin D,LDS751, and hydroxystilbamidine. All of the aforementioned nucleic acidstains are commercially available from suppliers such as MolecularProbes, Inc.

Still other examples of nucleic acid stains include the following dyesfrom Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green,SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1,LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3,TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3,PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II,SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24,-21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).

The detection system may be configured as a single molecule analysissystem (e.g., a single polymer analysis system). A single moleculedetection system is capable of analyzing single molecules separatelyfrom other molecules. Such a system may be capable of analyzing singlemolecules in a linear manner and/or in their totality. In certainembodiments in which detection is based predominately on the presence orabsence of a signal, linear analysis may not be required. However, thereare other embodiments embraced by the invention which would benefit fromthe ability to analyze linearly molecules (preferably nucleic acids) ina sample. These include applications in which the sequence of thenucleic acid is desired, or in which the polymers are distinguishedbased on spatial labeling pattern rather than a unique detectable label.

Thus, the polymers can be analyzed using linear polymer analysissystems. A linear polymer analysis system is a system that analyzespolymers such as nucleic acids, in a linear manner (i.e., starting atone location on the polymer and then proceeding linearly in eitherdirection therefrom). As a polymer is analyzed, the detectable labelsattached to it are detected in either a sequential or simultaneousmanner. When detected simultaneously, the signals usually form an imageof the polymer, from which distances between labels can be determined.When detected sequentially, the signals are viewed in histogram (signalintensity vs. time) that can then be translated into a map, withknowledge of the velocity of the polymer. It is to be understood that insome embodiments, the polymer is attached to a solid support, while inothers it is free flowing. In either case, the velocity of the polymeras it moves past, for example, an interaction station or a detector,will aid in determining the position of the labels relative to eachother and relative to other detectable markers that may be present onthe polymer.

An example of a suitable system is the GeneEngine™ (U.S. Genomics, Inc.,Woburn, Mass.). The Gene Engine™ system is described in PCT patentapplications WO98/35012 and WO00/09757, published on Aug. 13, 1998, andFeb. 24, 2000, respectively, and in issued U.S. Pat. No. 6,355,420 B1,issued Mar. 12, 2002. The contents of these applications and patent, aswell as those of other applications and patents, and references citedherein are incorporated by reference herein in their entirety. Thissystem is both a single molecule analysis system and a linear polymeranalysis system. It allows, for example, single nucleic acids to bepassed through an interaction station in a linear manner, whereby thenucleotides in the nucleic acid are interrogated individually in orderto determine whether there is a detectable label conjugated to thenucleic acid. Interrogation involves exposing the nucleic acid to anenergy source such as optical radiation of a set wavelength. Themechanism for signal emission and detection will depend on the type oflabel sought to be detected, as described herein.

The nature of such detection systems will depend upon the nature of thedetectable moiety used to label the polymer. The detection system can beselected from any number of detection systems known in the art. Theseinclude an electron spin resonance (ESR) detection system, a chargecoupled device (CCD) detection system, a fluorescent detection system,an electrical detection system, a photographic film detection system, achemiluminescent detection system, an enzyme detection system, an atomicforce microscopy (AFM) detection system, a scanning tunneling microscopy(STM) detection system, an optical detection system, a nuclear magneticresonance (NMR) detection system, a near field detection system, and atotal internal reflection (TIR) detection system, many of which areelectromagnetic detection systems.

Optical detectable signals are generated, detected and stored in adatabase. The signals can be analyzed to determine structuralinformation about the polymer. The signals can be analyzed by assessingthe intensity of the signal to determine structural information aboutthe polymer. The computer may be the same computer used to collect dataabout the polymers, or may be a separate computer dedicated to dataanalysis. A suitable computer system to implement embodiments of thepresent invention typically includes an output device which displaysinformation to a user, a main unit connected to the output device and aninput device which receives input from a user. The main unit generallyincludes a processor connected to a memory system via an interconnectionmechanism. The input device and output device also are connected to theprocessor and memory system via the interconnection mechanism. Computerprograms for data analysis of the detected signals are readily availablefrom CCD (Charge Coupled Device) manufacturers.

Other interactions involved in methods of the invention will produce anuclear radiation signal. As a radiolabel on a polymer passes throughthe defined region of detection, nuclear radiation is emitted, some ofwhich will pass through the defined region of radiation detection. Adetector of nuclear radiation is placed in proximity of the definedregion of radiation detection to capture emitted radiation signals. Manymethods of measuring nuclear radiation are known in the art includingcloud and bubble chamber devices, constant current ion chambers, pulsecounters, gas counters (i.e., Geiger-Müller counters), solid statedetectors (surface barrier detectors, lithium-drifted detectors,intrinsic germanium detectors), scintillation counters, Cerenkovdetectors, to name a few.

Other types of signals generated are well known in the art and have manydetections means which are known to those of skill in the art. Some ofthese include opposing electrodes, magnetic resonance, and piezoelectricscanning tips. Opposing nanoelectrodes can function by measurement ofcapacitance changes. Two opposing electrodes create an area of energystorage, located effectively between the two electrodes. It is knownthat the capacitance of such a device changes when different materialsare placed between the electrodes. This dielectric constant is a valueassociated with the amount of energy a particular material can store(i.e., its capacitance). Changes in the dielectric constant can bemeasured as a change in the voltage across the two electrodes. In thepresent example, different nucleotide bases or unit specific markers ofa polymer may give rise to different dielectric constants. Thecapacitance changes as the dielectric constant of the unit specificmarker of the polymer per the equation: C=KC_(o), where K is thedielectric constant and C_(o) is the capacitance in the absence of anybases. The voltage deflection of the nanoelectrodes is then outputted toa measuring device, recording changes in the signal with time.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the invention. The advantages and objects of the inventionare not necessarily encompassed by each embodiment of the invention.

1. A chip for use in detecting an agent, the chip comprising: a microfluidic channel incorporated into the chip, the microfluidic channel adapted to deliver a fluid that may contain an agent to a detection zone that lies at least partially in the channel; an illuminator incorporated into the chip, the illuminator positioned to direct an excitation signal to the detection zone; and a concave reflector incorporated into the chip and having a focal point substantially at the detection zone, the concave reflector positioned to receive an emission signal from the agent, when present in the detection zone, and to reflect the emission signal to a detector.
 2. The chip of claim 1, wherein the concave reflector is incorporated into the chip in a fixed relationship with respect to the channel.
 3. The chip of claim 2, further comprising: a solid medium that provides a pathway from the microfluidic channel to the concave reflector along which the emission signal can travel without substantial refraction.
 4. The chip of claim 3, wherein the solid medium extends from a wall of the channel to the concave reflector.
 5. The chip of claim 3, further comprising: a cover slip adapted to mate with the chip to enclose the channel, the concave reflector being incorporated into the cover slip.
 6. The chip of claim 5, wherein the cover slip and the solid medium each have a substantially similar refractive index.
 7. The chip of claim 5, wherein the solid medium provides a pathway from the microfluidic channel to the illuminator along which the excitation signal can travel without substantial refraction.
 8. The chip of claim 1, wherein the illuminator and the concave reflector are on opposed sides of the channel.
 9. The chip of claim 8, wherein the illuminator is a refractive illuminator having a focal point substantially located at the focal point of the concave reflector.
 10. The chip of claim 8, wherein the illuminator is a reflective illuminator having a focal point substantially located at the focal point of the concave reflector.
 11. The chip of claim 8, wherein the concave reflector includes an aperture positioned to allow the excitation signal to pass therethrough to prevent the excitation signal from being reflected toward the detector.
 12. The chip of claim 8, wherein the concave reflector is constructed and arranged to reflect at least a portion of the excitation signal.
 13. The chip of claim 12, further comprising: a wavelength specific filter positioned to receive the emission signal and the excitation signal from the concave reflector, the wavelength specific filter adapted to direct at least a portion of the emission signal to the detector and to prevent the excitation signal from reaching the detector.
 14. The chip of claim 12, further comprising: a second reflector constructed and arranged to receive and reflect the emission signal to the detector while allowing the excitation signal to pass thereby.
 15. The chip of claim 1, wherein the illuminator includes a refractive illuminator having a focal point substantially located at the focal point of the concave reflector and further wherein the illuminator and the concave reflector are positioned on a common side of the channel.
 16. The chip of claim 15, wherein the illuminator and the concave reflector are constructed and arranged such that overlap of the excitation signal and the emission signal is minimized.
 17. The chip of claim 16 wherein the concave reflector includes a central aperture and further wherein the refractive illuminator is positioned at the central aperture.
 18. The chip of claim 17, further comprising: a second reflector constructed and arranged to reflect the excitation signal back through the aperture and toward the refractive illuminator.
 19. The chip of claim 18, wherein the second reflector is substantially located at the focal point of the concave reflector.
 20. The chip of claim 17, further comprising: a second reflector constructed and arranged to reflect the excitation signal across the emission signal at a position outside of the chip.
 21. The chip of claim 1, wherein the concave reflector has a collection half angle greater than 49 degrees within water in a rectangular channel.
 22. The chip of claim 1, wherein the concave reflector has a collection half angle greater than 78 degrees within water in a rectangular channel.
 23. The chip of claim 1, wherein the concave reflector has a collection half angle of 90 degrees within water in a rectangular channel.
 24. The chip of claim 1, wherein the reflector has a collector numerical aperture of 1.0 or greater.
 25. The chip of claim 1, wherein the reflector has a collector numerical aperture of 1.3 or greater.
 26. The chip of claim 1, wherein the reflector is a parabolic reflector.
 27. The chip of claim 1, wherein the illuminator includes a waveguide incorporated into the chip.
 28. The chip of claim 27, wherein the waveguide is constructed and arranged to illuminate the microfluidic channel with an evanescent excitation signal.
 29. The chip of claim 1, comprising a plurality of pairs of concave reflectors and illuminators, each pair associated with a corresponding detection zone.
 30. The chip of claim 29, wherein the chip comprises more than 50 pairs of concave reflectors and illuminators.
 31. The chip of claim 29, wherein the plurality of pairs of concave reflectors and illuminators are arranged in serial along a common channel.
 32. The chip of claim 29, wherein the chip comprises a plurality of channels, wherein the plurality of pairs of concave reflectors and illuminators are arranged about the plurality of channels.
 33. The chip of claim 1, wherein the detection zone is circular in shape.
 34. The chip of claim 33, wherein the detection zone has a diameter of about 1.7 microns.
 35. The chip of claim 33, wherein the illuminator has an illuminator numerical aperture between 0.18 and 0.20.
 36. The chip of claim 1, wherein the detection zone is elliptical in shape.
 37. The chip of claim 1, wherein the elliptical detection zone has a minor diameter of about 1.7 microns.
 38. The chip of claim 1, wherein the detection zone extends beyond the channel.
 39. The chip of claim 1, wherein the agent comprises a plurality of agents.
 40. The chip of claim 1, wherein the agent is a polymer.
 41. The chip of claim 40, wherein the polymer is a nucleic acid optionally selected from the group consisting of DNA or RNA.
 42. The chip of claim 40, wherein the polymer is a peptide or a polypeptide.
 43. The chip of claim 1, wherein the agent is a cell.
 44. The chip of claim 1, wherein the agent is a pathogen.
 45. The chip of claim 1, further comprising: a registration feature for aligning the reflector relative to the detection zone. 46.-51. (canceled) 